AIDS is induced by the HIV-1 virus. HIV- 1 not only directly kills infected CD4(+) T cells, but also induces immunosuppression of uninfected T cells. Two immunosuppressive proteins, interferon alpha and extracellular Tat, mediate this process.
It is known that interferons exert a broad spectrum of biological activity such as anti-viral, anti-proliferative and immunomodulatory activities. As appreciated by those skilled in the art, interferon is a low-molecular weight polypeptide produced as an excretion from various types of mammalian cells. Its properties, chemical nature and methods of preparation have been extensively studied and documented. Currently, three major types of human interferons are known, each differentiated according to the initial producer cell and the inducer applied. They are interferons alpha (leukocyte), beta (fibroblast) and gamma (immune).
Interferon alpha was among the first of the cytokines to be produced by recombinant DNA technology and has been shown to have therapeutic value in conditions such as inflamrnatory, viral, and malignant diseases.
The HIV-1 virus is composed of several proteins. Among these proteins, the Tat and Nef proteins are the earliest produced within the viral cycle. As previously mentioned, extracellular Tat protein has immunosuppressive activity. The Tat protein is also an efficient activator of HIV gene expression.
Several approaches have been proposed to fight against the effects of Tat protein, such as genetic approaches to block the transcriptional action of Tat. Clinical trials have been performed with antisense RNA (Hybridon, Inc.), and in vitro experiments have shown the feasibility of ribozymes that use RNA of the TAR region (Tat binding site in RNA) to block the transcriptional action of Tat. Some pharmacological molecules inhibiting the transcriptional effect of Tat have also been described. All these methods were intended to block virus replication by blocking Tat-transcriptional activity.
Immunological methods have also been proposed. WO-A-91/18454 reports the utilization of retroviral protein polypeptides, including the native Tat protein, obtained by proteolytic degradation, chemical synthesis or genetic recombination, for specific immunizations. These retroviral protein polypeptides are used as immunogens to prevent retroviral replication and cytotoxic activity, particularly against the lymphocytes and neural cells affected by retroviral proteins like Tat. However, the toxicity of native proteins or their fragments precludes their utilization for immunization.
WO-A-95/31999 reports the utilization of the HIV-1 native Tat protein, fragments of the protein or polypeptides with deletions/substitutions, as immunogens to block HIV-1 replication by blocking the uptake of extracellular Tat protein by non-infected cells. In spite of the described immunogenicity of the compounds reported in WO-A-95/31999, certain concerns remain such as the toxic effects of native Tat protein, the immunological disorders induced by it, and the absence of neutralizing antibodies by immunization with the described immunogens. Therefore, there is a need for developing new Tat inactivated immunogens which have lost all the harmful effects of native Tat protein and which are also capable of inducing cellular and/or humoral immunological responses.
WO-A-96/27389 reports the utilization of Tat toxoids and retroviral regulatory proteins as immunogens capable of inducing an immunological response against the native Tat protein and preventing or repairing its immunosuppressive effects. These toxoids, inactive but immunogenic products, have been prepared by chemical treatment with formaldehyde of the native protein or segments derived from this protein. However, aldehyde inactivation is not readily reproducible. For industrial production, regular results are needed, especially regarding inactivation procedures. Constant structure and well-defined products are also preferred. An ideal product should also have a good stability period.